Tailoring of PEGylated bilosomes for promoting the transdermal delivery of olmesartan medoxomil: in-vitro characterization, ex-vivo permeation and in-vivo assessment

Figures & data

Table 1 The levels of independent variables and the model summary statistics of 2⁴ full factorial design used for optimization of OLM loaded PBs

Factors (independent variables)	Levels of variables
X1: BS type				SDC				STC
X2: EA type				Brijo20				Brij52
X3: BS amount (mg)				5				15
X4: EA amount (mg)				15				25
Responses (dependent variables)	R^2	Adjusted R^2	Predicted R^2	Constraints	p-value	F value	Adequate precision	Significant factors
Y1: EE%	0.97	0.96	0.95	Maximize	<0.0001	101.00	33.92	x1, x2, x3, x4
Y2: PS (nm)	0.74	0.62	0.41	Minimize	0.0002	6.20	9.11	x1, x2, x3, x4
Y3: PDI	0.27	−0.06	−0.67	Minimize	0.62	0.81	3.45	-
Y4: ZP (mV)	0.75	0.64	0.43	Maximize (as absolute value)	0.0002	6.55	9.21	x1, x2, x4
Y5: Q6h (%)	0.99	0.98	0.97	Maximize	<0.0001	223.74	48.49	x1, x2, x3, x4

Abbreviations: BS, bile salt; EA, edge activator; EE%, entrapment efficiency percent; PBs, PEGylated bilosomes; PS, particle size; PDI, polydispersity index; SDC, sodium deoxycholate; STC, sodium taurocholate; ZP, zeta potential; Q6h, amount of drug released after 6 hrs.

Table 2 A) Experimental runs, independent variables and measured responses of the 2⁴ full factorial experimental design of OLM PBs and B) the observed, predicted values and bias percent of the optimum PB15

A) Formulations	X1	X2	X3	X4	Y1 (%)	Y2 (nm)	Y3	Y4 (mV)	Y5 (%)
PB1	SDC	Brijo20	5	15	56.65±0.83	496.65±51.45	0.69±0.11	−32.90±1.60	91.98±0.45
PB2	SDC	Brijo20	5	25	53.14±0.45	420.90±16.30	0.46±0.04	−30.57±0.25	87.36±4.11
PB3	SDC	Brijo20	15	15	45.86±0.85	438.00±49.00	0.48±0.18	−33.60±0.40	91.93±1.44
PB4	SDC	Brijo20	15	25	40.19±0.12	387.35±48.45	0.52±0.08	−27.40±0.20	97.56±0.66
PB5	SDC	Brij52	5	15	72.65±0.43	562.10±7.90	0.37±0.07	−28.10±0.50	60.62±0.51
PB6	SDC	Brij52	5	25	59.73±0.24	543.15±23.15	0.61±0.11	−31.75±0.65	66.03±1.59
PB7	SDC	Brij52	15	15	47.87±0.13	545.30±5.30	0.52±0.03	−29.25±0.65	68.72±3.75
PB8	SDC	Brij52	15	25	43.82±0.18	440.80±69.20	0.63±0.04	−31.60±0.40	71.90±1.03
PB9	STC	Brijo20	5	15	76.90±0.38	616.15±37.75	0.55±0.15	−29.70±0.45	51.90±1.89
PB10	STC	Brijo20	5	25	70.82±0.16	464.65±23.25	0.50±0.12	−26.15±0.45	55.66±1.11
PB11	STC	Brijo20	15	15	68.00±1.38	561.40±1.20	0.49±0.09	−30.35±0.45	58.97±1.87
PB12	STC	Brijo20	15	25	60.68±0.30	440.30±24.30	0.60±0.03	−30.55±1.80	66.41±1.84
PB13	STC	Brij52	5	15	81.21±0.44	804.20±41.30	0.61±0.11	−44.50±0.25	47.18±1.62
PB14	STC	Brij52	5	25	78.11±0.20	646.75±24.25	0.63±0.04	−35.65±0.35	51.76±7.56
PB15	STC	Brij52	15	15	72.49±0.38	559.30±10.70	0.57±0.15	−38.35±0.65	59.60±0.24
PB16	STC	Brij52	15	25	69.47±0.12	492.30±54.30	0.50±0.14	−33.35±0.85	63.79±1.66
B)
Observed values of PB15					72.49	559.30	0.57	−38.35	59.60
Predicted values of PB15					72.33	606.26	0.56	−38.65	58.33
Bias %					0.22	8.39	1.75	0.78	2.17

Note: Data represented as mean±SD (n=3).

Abbreviations: X₁, BS type; X₂, EA type; X₃, BS amount; X₄, EA amount; Y₁ (EE%), entrapment efficiency percent (%); Y₂ (PS), particle size (nm); Y₃ (PDI), polydispersity index; Y₄ (ZP), zeta potential (mV); Y₅ (Q6h), amount of drug released after 6 hrs (%); PBs, PEGylated bilosome.

Figure 1 Response 3-D plots for the effect of BS type (X₁), EA type (X₂), BS amount (X₃) and EA amount (X₄) on EE%, PS and PDI of OLM loaded PBs.

Abbreviations: BS, bile salt; EA, edge activator; EE%, entrapment efficiency percent; PS, particle size; PDI, polydispersity index; OLM, olmesartan medoxomil; PBs, PEGylated bilosomes.

Figure 2 Response 3-D plots for the effect of BS type (X₁), EA type (X₂), BS amount (X₃) and EA amount (X₄) on ZP and Q6h of OLM loaded PBs.

Abbreviations: BS, bile salt; EA, edge activator; ZP, zeta potential; Q6h, amount of drug released after 6 hrs; OLM, olmesartan medoxomil; PBs, PEGylated bilosomes.

Figure 3 The release profiles of OLM from the prepared 16 PB formulations compared to OLM suspension (A) release profiles of OLM and PB1:PB8 and (B) release profiles of OLM and PB9:PB16. Data represented as mean±SD (n=3).

Abbreviations: OLM, olmesartan medoxomil; PB, PEGylated bilosome.

Figure 4 Morphology of the selected vesicle (PB15).

Abbreviation: PB, PEGylated bilosome.

Figure 5 DSC thermograms of (A) OLM, (B) cholesterol, (C) Span 60, (D) Brij52, (E) STC, (F) physical mixture of PB15 components and (G) PB15.

Abbreviations: STC, sodium taurocholate; OLM, olmesartan medoxomil; PB, PEGylated bilosome.

Table 3 Effect of storage on the physical properties of PB15

Parameter	PB15 fresh	PB15 after 45 days at 4°C	PB15 after 45 days at 25°C	PB15 after 90 days at 4°C	PB15 after 90 days at 25°C
EE%	72.49±0.38	72.04±0.95	70.52±3.91	68.33±3.44	67.93±5.75
PS (nm)	559.30±10.70	563.15±4.60	560.30±9.20	561.86±6.40	562.75±7.90
PDI	0.42±0.15	0.47±0.05	0.48±0.08	0.44±0.15	0.45±0.05
ZP (mV)	−38.35±0.65	−38.10±9.82	−36.35±8.52	−35.90±9.17	−34.40±6.89
Q6h (%)	59.60±0.34	61.60±1.00	63.36±1.88	62.48±3.93	63.12±3.02

Note: Data presented as mean±SD (n=3).

Abbreviations: EE%, entrapment efficiency percent; PS, particle size; PDI, polydispersity index; ZP, zeta potential; Q6h, amount of drug released after 6 hrs; PB, PEGylated bilosome.

Table 4 Skin permeability parameters of OLM after topical application of OLM suspension and PB15

Skin permeability parameters	OLM suspension	PB15
Total amount of OLM permeated per unit area after 12 h (μg/cm^2)	123.78±14.02	811.31±146.67
Jmax (μg/cm^2/h)	10.31±1.16	67.61±12.22
ER	1	6.56

Note: Data presented as mean±SD (n=3).

Abbreviations: J_max, permeation flux; ER, enhancement ratio; PB, PEGylated bilosome.

Figure 6 Cumulative amount of OLM permeated per unit area from PB15 relative to OLM suspension. Data represented as mean±SD (n=3).

Abbreviations: OLM, olmesartan medoxomil; PB, PEGylated bilosome.

Figure 7 A tile scan confocal laser microscope photomicrographs of longitudinal section in rat’s skin treated with (A) FDA control solution (B) fluoro-labeled PB15. (I) fluorescence light, (II) transmitted light and (III) merge between fluorescence light and transmitted light.

Abbreviations: FDA, fluorescein diacetate; E, epidermis; D, dermis; HF, hair follicles; PB, PEGylated bilosome.

Figure 8 Photomicrographs showing histopathological sections (hematoxylin and eosin stained) of normal untreated rat’s skin (group I), rat’s skin treated with OLM suspension (group II) and rat’s skin treated with PB15 (group III) with magnification power of 16x and 40x, respectively.

Abbreviations: E, epidermis; D, dermis; HF, hair follicles; OLM, olmesartan medoxomil; PB, PEGylated bilosome.

Figure 9 In-vivo skin deposition profiles of OLM versus time from PB15, transethosomes and OLM suspension after topical application. Data represented as mean±SD (n=3).

Abbreviations: OLM, olmesartan medoxomil; PB, PEGylated bilosome.

Table 5 Influence of oral tablets and PB15 on mean BP in methylprednisolone acetate-induced hypertensive rats

Time (hours)	Normal control (mmHg)	Hypertensive control (mmHg)	Oral tablets (mmHg)	PB15 (mmHg)	Percent reduction in BP of oral tablets	Percent reduction in BP of PB15
0	109.50±3.50	171.00±4.00	177.00±3.26	169.00±11.70	4.00±2.00%	6.00±4.00%
1	126.20±1.20	187.50±2.50	138.00±0.81	121.50±10.10	26.00±0.00%	34.00±2.00%
2	129.00±1.00	166.50±6.50	127.20±1.83	120.00±2.50	24.00±1.00%	28.00±2.00%
4	126.70±2.70	168.00±8.00	128.90±0.83	115.70±11.30	23.00±1.00%	31.00±7.00%
6	117.70±4.20	166.00±4.00	126.50±6.94	124.70±4.40	24.00±4.00%	25.00±3.00%
8	114.70±1.70	169.50±2.50	140.10±6.40	123.20±13.30	17.00±4.00%	27.00±8.00%
24	117.70±0.70	185.50±5.50	165.00±8.16	123.30±4.02	11.00±4.00%	35.00±5.00%
26	117.00±1.00	186.50±8.50	166.80±11.27	127.30±3.20	6.00±2.00%	24.00±2.00%
28	111.00±3.00	170.00±8.00	169.00±0.81	135.60±10.00	1.00±0.00%	20.00±6.00%
48	128.50±1.50	175.50±2.50	173.60±5.73	152.60±7.70	3.00±1.00%	13.00±4.00%

Note: Data presented as mean±SD (n=6).

Abbreviations: BP, Blood pressure; PB, PEGylated bilosome.

Table 6 Pharmacokinetic parameters of olmesartan after administration of oral tablets and PB15

Pharmacokinetic parameters	Oral tablets	PB15
Cmax (ng/mL)^a	270.08±79.51	296.52±177.47
Tmax (h)^b	1	7
AUC0→48 (ngh/mL)^a	1542.43±713.33	3625.61±948.02
AUC0→∞ (ngh/mL)^a	1583.41±676.84	3721.67±878.73
Relative bioavailability (%)	-	235.04

Notes: ^aData represented as mean±SD (n=6); ^bData represented as median (n=6).

Abbreviations: AUC, area under the curve; PB, PEGylated bilosome.

Figure 10 Pharmacokinetic profiles of olmesartan versus time from oral tablets and topically applied PB15. Data represented as mean±SD (n=6).

Abbreviation: PB, PEGylated bilosome.